A longstanding worldwide demand exists for new, effective, environmentally friendly, and safe means to control pests that damage agriculture or serve as disease vectors. Agriculture costs incurred by pests exceed billions of dollars annually in decreased crop yields, reduced crop quality, increased harvesting costs, pesticide application costs, and negative ecological impact. In addition to agriculture pests, many blood-feeding insects and cockroaches are vectors for pathogenic microorganisms that threaten human and animal health, or are annoying at the least. As in the case of agriculture pests, direct and intangible costs incurred by blood-feeding and household pests concern pesticide safety hazards to humans and animals, bioaccumulation and environmental incompatibility, and synthesis and application costs.
Almost all field crops, nursery and horticulture plants, and commercial farming areas are susceptible to attack by one or more pests. Particularly problematic are Coleopteran and Lepidopteran pests. An example of a Lepidopteran pest is the hornworm larva of Manduca sexta, and an example of a Coleopteran pest is the Colorado potato beetle, Leptinotarsa decemlineata. Vegetable and cole crops, lentils, leafy vegetables, melons, peppers, potatoes and related tubers, tomatoes, cucumbers and related vine crops, as well as a variety of spices are sensitive to infestation by one or more pests including loopers, armyworms, moth larvae, budworms, webworms, earworms, leafeaters, borers, cloverworms, melonworms, leafrollers, various caterpillars, fruitworms, hornworms, and pinworms. Likewise, pasture and hay crops such as alfalfa, pasture and forage grasses and silage are often attacked by a variety of pests including armyworms, alfalfa caterpillars, European skipper, a variety of loopers and webworms, as well as yellowstriped armyworms.
Fruit (including citrus), nut, and vine crops are susceptible to attack by a variety of pests, including sphinx moth larvae, cutworms, skippers, fireworms, leafrollers, cankerworms, fruitworms, girdlers, webworms, leaffolders, skeletonizers, shuckworms, hornworms, loopers, orangeworms, tortrix, twig borers, casebearers, spanworms, budworms, budmoths, and a variety of caterpillars and armyworms.
Field crops are targets for infestation by insects including armyworm, asian and other corn borers, a variety of moth and caterpillar larvae, bollworms, loopers, rootworms, leaf perforators, cloverworms, headworms, cabbageworms, leafrollers, podworms, cutworms, budworms, hornworms, and the like. Pests also frequently feed upon bedding plants, flowers, ornamentals, vegetables, container stock, forests, fruit, ornamental, shrubs and other nursery stock. Even turf grasses are attacked by a variety of pests including armyworms and sod webworms.
For the past 50 years growers, health officials, and the public have depended on chemical pesticides for controlling a variety of pests. However, environmental experts, health officials, and the public have become concerned about the amount of residual chemicals found in food, ground water, and elsewhere in the environment. Regulatory agencies around the world are restricting and/or banning the uses of many synthetic pesticides, particularly those that are persistent in the environment and that enter the food chain. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests. Some synthetic chemical pesticides poison the soil and underlying aquifers, pollute surface waters as a result of runoff, and destroy non-target life forms. These synthetic chemical pest control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They can also pose health hazards to the people applying them, especially if the proper application techniques are not followed.
Because crops of commercial interest are often the targets of pests, environmentally sensitive methods for controlling or eradicating pest infestations are desirable in many instances. This is particularly true for farmers, nurserymen, growers, and commercial and residential areas which seek to control pest populations using environmentally friendly compositions.
The most widely used environmentally friendly pesticidal formulations developed in recent years have been microbial pesticides derived from the bacterium Bacillus thuringiensis (“B.t.”). B. thuringiensis is a Gram-positive bacterium that produces proteins which are toxic to certain orders and species of pests. Many different strains of B.t. have been shown to produce insecticidal proteins. Compositions including B.t. strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are toxic to specific target pests, but are harmless to plants and other non-target organisms. The specificity of these toxins is often strain-specific, with certain toxins being active against a relatively narrow spectrum of pests. Indeed, many B.t. toxins have been identified that are active only against particular insect orders (e.g., dipterans, hymenopterans, coleopterans, etc.). This limitation prevents the use of a single B.t. endotoxin composition as a broad-range pesticide.
Crop pests are not the only targets for which an environmentally friendly and safe pesticide would be highly desirable. Many blood-feeding pests are known to prey on humans and animals, and many pests are vectors for pathogenic microorganisms that threaten human and animal health, including commercially important livestock, pets and other animals. The order Diptera contains a large number of blood-ingesting and disease-carrying pests, including, for example, mosquitoes, black flies, no-see-ums (punkies), horse flies, deer flies and tsetse flies.
Various species of mosquitoes, for example, transmit diseases caused by viruses, and many are vectors for disease-causing nematodes and protozoa. Mosquitoes of the genus Anopheles transmit Plasmodium, the protozoan that causes malaria. The mosquito species Aedes aegypti transmits an arbovirus that causes yellow fever in humans. Other viruses transmitted by Aedes species include the causative agents of dengue fever, eastern and western encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex, which includes the common house mosquito C. pipiens, is implicated in the transmission of various forms of encephalitis, filarial worms, and West Nile virus. Trypanasoma cruzi, the causative agent of Chagas disease, is transmitted by various species of blood ingesting Triatominae bugs. Tsetse flies (Glossina spp.) transmit African trypanosomal diseases of humans and cattle. Other diseases are transmitted by various blood-ingesting pest species.
Various pesticides have been employed in efforts to control or eradicate populations of disease-transmitting pests. For example, DDT, a chlorinated hydrocarbon, has been used in attempts to eradicate malaria-bearing mosquitoes throughout the world. Other examples of chlorinated hydrocarbons are BHC, lindane, chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin, dieldrin, chlordane, heptachlor, and endrin). The long-term stability of many of these pesticides and their tendency to bioaccumulate render them particularly dangerous to many non-target organisms.
In addition to environmental concerns, another major problem associated with conventional chemical control practices is the capability of many species to develop pesticide resistance. Resistance results from the selection of naturally occurring mutants possessing biochemical, physiological or behavioral factors that enable the pests to tolerate the pesticide when it is applied.
There is clearly a longstanding need in the art for pesticidal compounds that reduce or eliminate direct and/or indirect threats to human health posed by presently available pesticides, that are environmentally compatible and safe, are not toxic to non-pest organisms, and have a reduced tendency to bioaccumulate.
Approaches to pesticide development are lacking that involve specifically disrupting key pest regulatory processes, notably membrane organic solute transporters, organic solute- and amino acid-gated ion channel proteins, and/or amino acid metabolic pathways as targets. The development of such methodologies could provide safer, environmentally friendly alternatives to conventional commercially used pesticides, and provide more economical means for suppressing or eradicating target pest populations. The formulation of pesticidal compositions that are non-toxic to animals and to humans would greatly enhance the present methods available for killing pests, and would provide alternative strategies for environmentally responsible pest management.
Membrane organic solute transporter proteins and ion channel proteins serve critical roles in maintaining organic solute and ionic metabolic, thermodynamic, and electrical events in cells. In both eukaryotes and prokaryotes these proteins affect electrochemical gradients of a wide variety of metabolic molecules and electrolytes, including amino acids and related metabolites as well as H+, OH−, Na+, K+, Cl−, and carbonate ions (Gerencser and Stevens, 1994 J. Exper. Biol. 196:59–75; Stevens, B. R. 2001. “Theory and methods in nutrient membrane transport.” In: Surgical Research. pp. 845–856. W. W. Souba and D. W. Wilmore, eds., Academic Press, San Diego). Molecular cloning studies have identified several subfamilies of organic solute transporters and ion channels (Griffith, J. K. and C. E. Sansom, 1998, In: The Transporter Facts Book, Academic Press, San Diego, pp. 500).
Organic solute transporters and ion channels are commonly defined by their substrate selectivity within polypeptide superfamilies. For cloned or native secondary active transporters, it is generally assumed that cell membranes utilize ion and organic molecule electrochemical gradients to aid in exchanging these solutes between the cell interior and extracellular environment (Gerencser, G. A. and B. R. Stevens, 1994, J. Exper. Biol. 196:59–75; Stevens, B. R. 1999, Digestion and Absorption of Protein. In: Biochemical and Physiological Aspects of Human Nutrition. pp. 107–123, M. H. Stipanuk, ed., W.B. Saunders Co., Philadelphia). In the ‘prototypical’ transporter, organic solutes that can be moved across cell membranes by uniport, hetero- or homo-exchange, and/or uptake can be activated by ions, and/or thermodynamiclly cotransported with ions (cf refs in Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276(36):33413–33418; Griffith, J. K. and C. E. Sansom, 1998, In: The Transporter Facts Book, Academic Press, San Diego, pp. 500). Ion channels, on the other hand, are typically distinct from organic solute transporters, are selective in their conducting ion species, and may be gated by organic ligands (Hille, B, 2001, Ionic channels of excitable membranes, 3rd Edition, Sinauer Associates, Inc., Sunderland, Mass., pp. 814; Saier, M. H., 2000, Families of proteins forming transmembrane channels. J. Membrane Biol. 175:165–180). In some cases, multimeric (e.g., heterodimer, homodimer) interactions or associations among or between several polypeptides are responsible for the physiological activity of solute transport or ion conductance, or multifunctional solute transport/ion channel activity (Saier, M. H., 2000, A functional-phylogenetic classification system for transmembrane solute transporters, Micro. Molec. Biol. Rev 64:354–411; Verrey, F., Jack, D. L., Paulsen, I. T., Saier, M. H., and Pfeiffer, R., 1999, New glycoprotein-associated amino acid transporters, J. Membrane Biol., 172:181–192; Saier, M. H., 2000, Families of transmembrane transporters selective for amino acids and their derivatives, Microbiology, 146:1775–1795; Wagner, C. A., Lang, F., and Broer, S., 2001, Function and structure of heterodimeric amino acid transporters, Am. J. Physiol. Cell Physiol., 281:C1077–C1093; Kanai, Y., and Endou, H., 2001, Heterodimeric amino acid transporters: molecular biology and pathological and pharmcological relevance, Curr. Drug Metabol., 2:339–354; Saier, M. H., 2000, Families of proteins forming transmembrane channels. J. Membrane Biol. 175:165–180). The amino acid/polyamine/choline (APC) superfamily of transporters represents examples of polypeptides responsible for amino acid transport. Within the APC superfamily, a subunit family of polypeptide light chains (e.g., LAT polypeptides) require association with an ancillary heavy chain polypeptide subunit of the 4F2hc/CD98 family, for functional membrane transport activity (Saier, M. H., 2000, A functional-phylogenetic classification system for transmembrane solute transporters, Micro. Molec. Biol. Rev 64:354–411; Verrey, F., Jack, D. L., Paulsen, I. T., Saier, M. H., and Pfeiffer, R., 1999, New glycoprotein-associated amino acid transporters, J. Membrane Biol., 172:181–192; Wagner, C. A., Lang, F., and Broer, S., 2001, Function and structure of heterodimeric amino acid transporters, Am. J. Physiol. Cell Physiol,. 281:C1077–C1093; Kanai, Y., and Endou, H., 2001, Heterodimeric amino acid transporters: molecular biology and pathological and pharmcological relevance, Curr. Drug Metabol., 2:339–354).
Manduca sexta is a major crop pest whose larval stage, commonly known as tobacco hornworms, rapidly attacks and defoliates tobacco and tomato plants; the large fifth instar larvae are especially damaging. Other vegetable crops such as peppers, eggplant, and potatoes also can be affected. Tobacco and tomato hornworms rapidly grow and gain weight as they progress from the first instar stage (about 6.7 mm) through the fifth instar (about 81.3 mm) over a period of about 20 days. The sated larvae then drop to the soil to pupate, and eventually emerge as adult moths. The moths lay eggs, which develop into larvae, and the life cycle continues, thereby sustaining crop damage. Killing the larvae prevents immediate crop damage and prevents or reduces future damage by interrupting the life cycle.
The midgut region of Manduca sexta larvae displays compartments with the property of high concentrations of primarily K+ in an alkaline fluid (˜pH 10), with trans-epithelial potentials ˜250 mV (Harvey et al., 1999, Am. Zol. 38:426–441; Harvey and Wieczorek, 1997, J. Exper. Biol. 200:203–216). Epithelial cells of this region transport a variety of organic and inorganic solutes and nutrients, including amino acids and electrolytes, as demonstrated by in vitro isolated membrane vesicle uptake studies. In place of a Na+/K+-ATPase typically found in cells, this tissue instead possesses a proton translocating V-ATPase (Graf et al., 1992, FEBS Lett. 300:119–122; Merzendorfer et al. 1997, J. Exper. Biol. 200:225–235) which energizes the cell membranes for secretion and absorption of ions, primarily K+ and Na+, and establishment of a large pH gradient. A K+-activated leucine-preferring transporter (KAAT1) has been identified from the hornworm midgut (Castagna et al., 1998, Proc Natl. Acad. Sci. USA 95:5395–5400), and a GABA (gamma aminobutyric acid) transporter has been cloned from an Manduca sexta embryo cDNA library (Mbungu et al. 1995, Arch. Biochem. Biphys. 318:489–497).
CAATCH1 (Cation-Amino Acid Transporter/CHannel) is a recently cloned insect membrane protein initially cloned from Manduca Sexta. CAATCH1 exhibits a unique polypeptide and nucleotide sequence related to, but different from, mammalian Na+-, Cl−-coupled neurotransmitter transporters (Feldman et al., 2000, J. Biol. Chem. 275:24518–24526). Utilizing a unique PCR-based strategy, the gene encoding CAATCH1 was cloned (Feldman et al., 2000, supra) from a cDNA library in LambdaZap plasmids, obtained from the digestive midgut of Manduca sexta larvae.
The unanticipated and novel biochemical, physiological, and molecular properties of CAATCH1 indicated that it is a multi-function protein that mediates amino acid uptake in a manner that is thermodynamically uncoupled from ion electrochemical potentials, and furthermore that CAATCH1 simultaneously functions as an amino acid-modulated gated alkali cation channel (Quick, M. and B. R. Stevens, 2001, “Amino acid transporter CAATCH1 is also an amino acid-gated cation channel”. J. Biol. Chem 276:33413–33418) serving at least Na+ and K+. Radiotracer and electrophysiology experiments with functional CAATCH1 polypeptide expressed from the full length CAATCH1 cDNA demonstrated direct amino acid ligand-protein interactions, and indicated that binding by different amino acid substrates differentially affects the conformational states of CAATCH1 (Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276:33413–33418). Notably, L-methionine binding to CAATCH1 in situ in biomembranes in the presence of Na+ perturbs the charge-voltage relation with a high affinity binding constant, affecting transient currents due to CAATCH1-associated charge transfer across the membrane dielectric field. Furthermore, CAATCH1-associated voltage-dependent amino acid-elicited steady state inward cation currents are blocked by methionine, and indeed methionine reversed charge movements via CAATCH1 expressed in cell membranes, even though radiotracer methionine influx is catalyzed by CAATCH1 (Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276:33413–33418). In insects, CAATCH1 likely plays a broader role than that of a ‘classical’ transporter or channel; as a multifunction protein CAATCH1 is likely a key protein in electrolyte and organic solute homeostasis of certain insects (Feldman et al, 2000, J. Biol. Chem. 275(32):24518–24526)
Many pests—including mosquitoes, black flies, cockroaches, Lepidopterans, and Coleopterans—possess an alkaline pH midgut, and share some similar physiological mechanisms that occur within this unusual milieu (Nation, J., 2001, In: Insect Physiology and Biochemistry, CRC Press, Boca Raton, pp 496). Mosquito larvae possess such an alkaline midgut, and adjust free amino acid concentrations in their hemolymph and extracellular compartments in direct response to existence of foreign factors in the gut (e.g., B.t. δ-endotoxin) or the salinity of their habitat (Bounias, M. et al., 1989, J. Invertebr Pathol. 54:16–22). Notably, one standout free amino acid, L-proline, accumulates 4-fold during the normal course of Aedes aegypti larval development, and in Culex spp. L-proline accumulation can exceed 50-fold (up to 70 mM) when larvae are stimulated by Na+ in their feeding pools (Bounias, M. et al., 1989, supra; Patrick, M. L. and T. J. Bradley, 2000, J. Exp. Biol. 203:831–839; Chaput, R. L. and J. N. Liles, 1969, Ann. Entomol. Soc. Am. 62:742–747). This proline is likely utilized as an energy source and for osmoregulation (Patrick, M. L. and T. J. Bradley, 2000, J. Exp. Biol. 203:831–839; Bounias et al., 1989, J. Invertebr Pathol. 54:16–22). In contrast, free L-methionine has the distinction of virtually the lowest measurable concentration (≦0.001 mM) of any of the free amino acids in mosquito larvae. Virtually all methionine existing in the free amino acid state in larvae (Dadd, R. H., 1973, Ann Rev Entomol 18:381–420) is metabolically shunted and sequestered into so-called methionine-rich hexamerin proteins (Korochkina et al., 1997, Insect Biochem Mol. Biol. 27:813–824) that are stored for post-larval developmental events. Methionine analogues have been shown to modify metabolism in Lepidopterans and other insects (Rock, G. C. et al., 1973, Utilization of methionine analogues by Argyrotaenia velutinana Larvae. Ann Entomol Soc Am. 66:177–179). In its role as a nutrient transporter, the CAATCH1 protein has been shown to be primarily responsible for proline uptake (Feldman et al., 2000, J. Biol. Chem. 275(32):24518–24526), while in the presence of extremely low concentrations of methionine, CAATCH1 effectively shuts down ionic fluxes via its channel properties (Feldman et al., 2000, J. Biol. Chem. 275(32):24518–24526; Quick, M. and B. R. Stevens, 2001, J. Biol. Chem. 276(36):33413–33418).
Compared to conventional organic pesticides, the use of biodegradable small molecules, such as amino acids, as pesticides is highly desirable, owing to the safety of such compounds to humans, animals, and the environment.